ER-export and ARFRP1/AP-1–dependent delivery of SARS-CoV-2 Envelope to lysosomes controls late stages of viral replication

The β-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release, and pathogenesis. We developed a nondisruptive tagging strategy for SARS-CoV-2 E and find that, at steady state, it localizes to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae, responsible for endoplasmic reticulum–to–Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify an ADP ribosylation factor 1/adaptor protein–1 (ARFRP1/AP-1)–dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across β-coronaviruses, and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and, by developing a trans-complementation assay for SARS-CoV-2 structural proteins, that lysosomal delivery of E and its viroporin activity is necessary for efficient viral replication and release.


Fig. S2. Alanine-scanning mutagenesis of the cytoplasmic tail of E. (A)
Schematic of alanine-scanning mutagenesis of the C-terminus of E. In mutants M1 -M9, this indicated amino acids were exchanged for Alanine.(B) Representative images of VeroE6 cells expressing the indicated versions of E-mEmerald and LAMP1-tdTomato from at least 15 imaged cells per condition.(C) Quantification of E-mEmerald retention of in Golgiderived tubules in VeroE6 cells transfected with the indicated Alanine-scanning mutants from Fig. S2B.Quantification is from at least 15 imaged cells per experiment from N = 3 independent experiments.Mean ± S.E. is displayed, with statistical significance determined by one-way ANOVA with Dunnett's correction.(D) Mander's colocalization co-efficient of E-mEmerald expressing VeroE6 cells stained with antisera raised against LAMP1.Mean ± S.D. of E-mEmerald signal in LAMP1 positive lysosomes provided from 10 imaged cells.Significance calculated by 1-way ANOVA with Tukey's correction.(E) A549, Caco-2 or Calu-3 cells were transfected with plasmids encoding the indicated E-mEmerald, E-mEmerald M5 or E-mEmerald M9 images representative of between 39 and 60 imaged cells (A549); 31 and 38 imaged cells (Caco-2); 7 and 11 imaged cells (Calu-3).In microscopy panels, scale bars are 10 µm.Green HaloTag ligand (green), released into dye-free media in the presence or absence of 4-PBA for 6 hours and then re-stained with TMR HaloTag ligand to illuminate newly synthesised E-HT (magenta).Images representative of 50 imaged cells per experiment across 3 independent experiments.The newly synthesized E-HT images from this panel are additionally presented in Fig. 2J.In microscopy panels, scale bars are 10 µm.

Fig. S9. The role of ARFRP1 in coordinating AP-1 at the Golgi for Golgi-to-lysosome trafficking of E. (A)
Volcano plot depicting proteins recovered from neutravidin down from 293T cells expressing either HA-TurboID or E-HA/TurboID and subject to a 20-minute biotinylation.N = 3.All adaptor proteins (AP) identified are highlighted, with colours corresponding to the different AP complexes.Percentages were counted from proteins that changed abundance by more than 2-fold and were statistically significant at p < 0.05, as determined by FDR-corrected two-tailed T-tests.(B) VeroE6 cells, or VeroE6 cells transfected with E-mEmerald, were fixed and stained with antisera raised against AP1G1 or ARFRP1 as indicated.Images representative of 12 or 14 imaged cells respectively.(C) Resolved cell lysates from VeroE6 cells that had transfected with control siRNA or siRNA targeting AP1M1, AP1AR or GGA1 were examined by western blotting with antisera raised against AP1M1, AP1AR, GGA1 or GAPDH.(D, E) Representative images of WT or ARFRP1 -/-VeroE6 cells transfected with the indicated siRNA and a plasmid encoding E-mEmerald (D) with quantification of the number of cells displaying E-mEmerald tubules reported (E).15 cells per experiment were scored from N = 3 independent experiments.Significance calculated by one-way ANOVA comparing WT control against WT AP1M1 siRNA, WT control against GGA1 siRNA, and WT AP1AR siRNA against ARFRP1 -/-AP1AR siRNA, with Šidák's correction for multiple testing.(F) A549 cells were transfected with control siRNA or siRNA targeting AP1M1 and a plasmid encoding E-mEmerald.Cells were fixed and stained with antisera raised against LAMP1, GM130 or GRASP55 as indicated.Images representative of between 5 and 14 imaged cells per condition.In microscopy panels, scale bars are 10 µm.and specificity in VeroE6 cells.VeroE6 were transfected with siRNAs and either the E-subgenomic/E-genomic dual luciferase reporter, or N-subgenomic/E-genomic dual luciferase reporter, as above.Dual luminescence was recorded as described above.Data plotted shows the fold change of the ratio between E-sgmRNA reporter or N-sgmRNA reporter and E-genomic reporter for E-sgmRNA siRNA treatments, relative to a scrambled control siRNA.Mean ± S.E. is displayed, with N = 3, with statistical testing using a 2-way ANOVA using the Šidák correction.Relative depletion of E-sgmRNA or N-sgmRNA reported as > 1. Potent depletion of N-sgmRNA was obtained with siRNA 15 and 16, values for which (15 = 47.54 ; 16 = 34.86)are omitted from the plot.(E) VeroE6 cells were treated with 10 pmol E-sgmRNA targeting siRNA-9, or scrambled siRNA for 20 hours before being infected with SARS-CoV-2 (hCoV-19/England/02/2020) at 1 PFU/cell for 2 hours.After 24 hours, reverse transcription was performed and cDNA levels of SARS-CoV-2 ORF1ab, E and N sgmRNAs were quantified.The data plotted shows viral mRNA expression normalised to actin plotted relative to normalised ORF1a/b expression with the scrambled siRNA treatment.Mean ± S.E. is displayed, with N equal to the number of data points displayed.(F) Cartoon of pSBTet-RN.(G) VeroE6 cells constitutively expressing tdTomato and expressing a doxycycline-inducible E-mEmerald from the Sleeping Beauty retrotransposition vector were treated with doxycycline and imaged.Images representative of 5 imaged fields of view.(H) SARS-CoV-2 viruses from VeroE6 cells treated with either scrambled siRNA or E-sgmRNA targeting or siRNA-9 were generated.A diluted stock was applied to confluent VeroE6 cells for 30 minutes.Infected cells were grown for 3 days before being fixed and stained with 0.2% toluidine blue.Images show representative plaque formation.(I) Quantification of plaque formation represented as titre (PFU/ml) using the ViralPlaque FIJI macro.Mean ± S.E.presented from N = 8, with significance calculated by a 1-way ANOVA with Dunnet's correction comparing scrambled to E-sgmRNA siRNAs.(J) Quantification of the percentage of large plaques vs total plaques from plaque assays.Large plaques were defined by having an area greater than 0.82 mm 2 and measured using the ViralPlaque macro in FIJI.Mean ± S.E.presented from N = 8, with significance calculated by a 1-way ANOVA comparing scrambled to E-sgmRNA siRNAs with Dunnet's correction.In microscopy panels, scale bars are 10 µm.

Fig. S3 .
Fig. S3.The C-terminus of coronaviral E proteins encodes an ER-export motif (A, B) Representative images and quilt quantification from 50 imaged VeroE6 cells per condition transfected with a panel of E-mEmerald mutants in which the putative C-terminal PDZ-ligand in E (DLLV) was replaced by PDZ-ligands of different classes, or with chimaeric sequences from Middle East Respiratory Virus (MERS)-CoV or Murine Hepatitis Virus (MHV)-CoV (Strain S).The class of PDZ ligand is indicated, with Class I defined by -X-[S/T]-X-ф, Class II defined by -X-ф-X-ф and Class III defined by -X-[D/E/K/R]-X-ф (54) (C, D) Representative images and quilt quantification from 50 imaged VeroE6 cells per condition transfected with a panel of E-mEmerald mutants in which residues in the C-terminal ER-export motif (DLLV) were sequentially deleted or were replaced by Cterminal hydrophobic (AAAV) or di-hydrophobic (AALL) sequences.(E, F) Representative images of VeroE6 Fig. S5.(D) Hierarchical clustering performed on the median adjusted IBAQ values of each proteomic sample calculated and plotted by average Euclidean distance.Colour scale indicates protein abundance as measured by log2 transformed iBAQ values.(E) Volcano plot depicting proteins recovered from neutravidin pulldown from 293T cells expressing TurboID or E-HA/TurboID fusions and subject to a 20-minute biotinylation prior to lysis.Volcano plot constructed from N = 3 biological repeats.A selection of proteins strongly enriched by E-HA/TurboID is displayed in red.Percentages were counted from proteins that changed abundance by more than 2-fold and were statistically significant at p < 0.05, as determined by FDR-corrected two-tailed T-tests.(F) Cell lysates and HaloTrap-captured fractions from 293T cells transfected with the indicated E-HT Site3 fusions were resolved by SDS-PAGE and examined by western blotting with the indicated antisera (N = 3).

Fig. S5 .
Fig. S5.Comparative proximity biotinylation of ER-export proficient and ER-export defective versions of E. (A) Hierarchical clustering performed on the median adjusted IBAQ values of each proteomic sample calculated and plotted by average Euclidean distance.Colour scale indicates protein abundance as measured by log2 transformed iBAQ values.(B) Principal component analysis (PCA) on median averaged data across the three repeats for each sample.Red dots/text indicate WT-like E proteins, green dots/text indicates ΔDLLV-like E proteins, and grey indicates the cytoplasmic control.(C) Tabular depiction of PDZ-domain containing proteins recovered from comparative proteomics of WT and DDLLV versions of E-HA/TurboID.(D) Volcano plots depicting proteins recovered from a neutravidin pull down from 293T cells expressing either E-HA/TurboID Site3or E-HA/TurboID Site3 ΔDLLV, and subject to a 20-minute biotinylation.N = 3. Gene Ontology was used to assign recovered proteins to subcellular localisations.PDZ-domain containing proteins were annotated on the volcano plot.Percentages were counted from proteins that changed abundance by more than 2-fold and were statistically significant at p < 0.05, as determined by FDR-corrected two-tailed T-tests.

Fig. S6 .
Fig. S6.Investigation of Golgi-to-lysosome trafficking of SARS-CoV-2 E. (A).Representative images of VeroE6 cells transfected for 18 hours with plasmids encoding E-mEmerald and either an empty vector or HA-Dynamin2 K44A and then incubated with Alexa 647 -labelled Transferrin for 2 minutes.(B) Quantification of Transferrin puncta in cells from A, mean ± S.D. displayed, statistical significance determined by 2-tailed T-Test from 27 imaged cells per condition.Quantification of E-mEmerald puncta in cells from A. Mean ± S.D. displayed with statistical significance determined by 2-tailed T-Test from 25 imaged cells.(C, D) Volcano plot depicting proteins recovered from a neutravidin pulldown from 293T cells expressing HA/TurboID (cyto) or E-HA/TurboID (WT) fusions and subject to a 20-minute biotinylation prior to lysis.Volcano plot constructed from N = 3 biological repeats and normalized against cytoplasmic TurboID (C) or E-HA/TurboID DDLLV (D).Percentages were counted from proteins that changed abundance by more than 2-fold and were statistically significant at p < 0.05, as determined by FDR-corrected two-tailed T-tests.The position of 12 significantly enriched membrane trafficking proteins (fold change > 6 and -log10 based p-values of > 3; GORASP2 and SCAMP1 were additionally selected due to their extreme placement in terms of significance or fold change) selected for our knockout screen are depicted in red.The ARFRP1-related GTPase, ARF1, is highlighted in blue and was not significantly enriched.(E) Correlation plot of candidates returned by E-HA/TurboID Site3 vs E-HA/TurboID Site4 .In microscopy panels, scale bars are 10 µm.

Fig. S7 .
Fig. S7.Examination of E-mEmerald trafficking in a panel of knockout VeroE6 cells.(A) Genomic locus of the gRNA-annealing region and protospacer adjacent motif (PAM) of each target, and Next Generation Sequencing of allele-specific indels in edited VeroE6 clones, including allele percentages of the two most common reads.(B) Resolved lysates from WT or RER1 -/-VeroE6 cells were examined by western blotting with antisera raised against RER1 or GAPDH.(C, D) Representative images and quantification of subcellular distribution of the indicated knockout VeroE6 cells transfected with a plasmid encoding E-mEmerald.Quantification performed from 50 cells per condition.(E) VeroE6 cells expressing E-mEmerald and stained against ARFRP1 and the amount of E-mEmerald in ARFRP1-positive membranes was calculated by Mander's colocalisation coefficient (0.379 ± 0.106, mean ± S.D. from 26 imaged cells).In microscopy panels, scale bars are 10 µm.

Fig
Fig. S8.E-mEmerald is retained in Golgi-derived tubules in the absence of ARFRP1.(A) WT or ARFRP1 - /-VeroE6 cells were transfected with E-mEmerald, fixed and stained with antisera raised against LAMP1, GM130 or GRASP55.Images representative of between 7 and 26 imaged cells per condition.(B) Mander's coefficient, mean ± S.D. of E-mEmerald signal in LAMP1 positive lysosomes provided from 10 imaged cells.Significance calculated by an unpaired 2-tailed T-test.(C) Clustal Omega alignment of ARF1 and ARFRP1 with residues involved in GTPase activity (ARF1: Q71, T31; ARFRP1: Q79, T31) in red and residues involved in the hydrophobic effector patch (ARF1 Y81; ARFRP1 Y89) in green.(D) Representative images of ARFRP1 -/- VeroE6 cells with the single plasmid transfections as indicated and stained with antisera against ARFRP1.Images representative of between 10 and 15 imaged cells per condition.In microscopy panels, scale bars are 10 µm.

Fig. S10 .
Fig. S10.The b-coronavirus specific insertion binds AP-1.(A) Resolved cell lysates from WT or ARFRP1 -/- VeroE6 cells were examined by western blotting with antisera raised against GAPDH, AP1G1 or AP1M1.(B, C) VeroE6 cells were transfected with plasmids encoding the indicated ARFRP1 proteins and stained with antisera raised against ARFRP1 or AP1G1 (B).Transfected cells indicated by asterisks.Perinuclear localisation of AP1G1 was scored in the accompanying quilt (C) from 50 imaged cells per condition.Acquisition settings were optimised for overexpressed ARFRP1 staining.(D) Cell lysates and GFP-Trap immunoprecipitations of 293T cells co-transfected with plasmids encoding GFP or the indicated E-mEmerald constructs and HA-AP1B1 were resolved by SDS-PAGE and examined by western blotting with antisera raised against HA or GFP.(E) Quantification of data from D from N = 4 to 7 independent experiments as indicted, significance calculated by 1-way ANOVA with Dunnet's correction.In microscopy panels, scale bars are 10 µm.

Fig. S11 .
Fig. S11.Validation of siRNA designed to target SARS-CoV-2 Envelope sgmRNA.(A) Schematic showing the design of a dual-luciferase assay reporter to test siRNA efficacy and specificity against E subgenomic mRNA (sgmRNA).TRS elements and the annealing region of the E-sgmRNAs are labelled.(B) Schematic showing the design of E-sgmRNA targeting siRNAs relative to E-sgmRNA.(C) For initial screening, Hek293 cells were transfected with 20 µM of control or E-sgmRNA targeting siRNAs and after 24 hours were transfected with a 1:1 ratio of E-subgenomic/E-genomic dual-luciferase assay reporter plasmids.After 24 hours, luminescence of Firefly and Renilla luciferases was assessed.The ratio of E-sgmRNA reporter and E-genomic reporter was calculated and a fold change relative to scrambled siRNA plotted.Mean ± S.E. is displayed, with N = 3.Relative depletion of E-sgmRNA reported as >1.(D) Promising siRNAs from Hek293 cells (C) were tested for efficiency